Neurons and other excitable cells use ion channel proteins to generate electrical and chemical signals. Understanding the structure and functional mechanisms of voltage-activated ion channels is of particular significance because these proteins generate nerve impulses, providing a critical solution to the biological problem of signaling rapidly over long distances. A mechanistic understanding of these proteins is also of medical significance because they are involved in many disease, and are widely targeted by therapeutic drugs. Recent X-ray structures of voltage-activated potassium (Kv) channels have led to new ideas about how interactions between voltage-activated ion channels and the surrounding membrane are crucial for function of these channels. We have began exploring whether the new ideas about voltage sensors in Kv channels could be extended to voltage-activated sodium (Nav) channels. Eukaryotic Nav channels have an architecture that is similar to Kv channels, except that the channel forming alpha-subunits are much larger because they contain four repeating S1-S6 motifs, each resembling a single Kv subunit. One of the fascinating features of Nav channels is that the four distinct S1-S4 voltage-sensing domains appear to make unique contributions to the gating and pharmacological properties of the channel. The first three S1-S4 domains are thought to be crucial for opening Nav channels, for example, whereas the fourth plays an important role in inactivation. Given their pivotal role in rapid electrical signaling, it is not surprising that Nav channel are heavily targeted by toxins from venomous animals. Although several of these toxins have been shown to interact with specific S1-S4 domains, in most cases their receptors had not been identified and the possibility of a toxin interacting with multiple domains had not been explored. Taking advantage of the portable nature of paddle motifs, we were able to transplant S3b-S4 paddle motifs from Nav channels into Kv channels, and show that scorpion and tarantula toxins can interact with each of the four motifs in Nav channels, and that in many instances toxins interact with more than one voltage sensor. Our understanding of how a toxin interacting with a specific voltage sensor alters gating of the channel, however, remains incomplete because many toxins target multiple voltage sensors, and the effects of toxins on the gating of Nav/Kv chimeras are often distinct from those observed in Nav channels. We have identified an unusually toxin-insensitive Nav channel subtype that provides a foundation for transferring toxin-sensitivity to individual voltage sensors within the framework of an Nav channel. We propose to study how toxins interacting with individual voltage sensors alter the gating of Nav channels. The alpha-subunits of Nav channels do not exist alone in neurons, but are typically found together with beta-subunits, a family of single TM auxillary subunits that can dramatically alter the kinetics of inactivation. If voltage sensors interact in crucial ways with the surrounding membrane, the protein-lipid interface would be an interesting location for auxillary subunits to interact with specific voltage sensors and modulate the gating properties of the channel. In exploratory preliminary experiments we found that the beta1-subunit has large effects on movement of the voltage sensors in the brain Nav1.2 channel, and we found that another Nav channel variant is insensitive to the beta1-subunit. We propose to investigate how the auxillary subunit modulates the function and pharmacology of Nav channel voltage sensors, and to define where physical interactions occur.